The genome of an Escherichia coli MC4100 strain with a placMu50 fusion revealed numerous regulatory differences from MG1655, including one that arose during laboratory storage. The 194 mutational differences between MC4100(MuLac) and other K-12 sequences were mostly allocated to specific lineages, indicating the considerable mutational divergence between K-12 strains.

Eukaryotic initiator proteins form origin recognition complexes (ORCs) that bind to replication origins during most of the cell cycle and direct assembly of prereplication complexes (pre-RCs) before the onset of S phase. In the eubacterium Escherichia coli, there is a temporally similar nucleoprotein complex comprising the initiator protein DnaA bound to three high-affinity recognition sites in the unique origin of replication, oriC. At the time of initiation, this high-affinity DnaA-oriC complex (the bacterial ORC) accumulates additional DnaA that interacts with lower-affinity sites in oriC, forming a pre-RC. In this paper, we investigate the functional role of the bacterial ORC and examine whether it mediates low-affinity DnaAoriC interactions during pre-RC assembly. We report that E. coli ORC is essential for DnaA occupation of low-affinity sites. The assistance given by ORC is directed primarily to proximal weak sites and requires oligomerization-proficient DnaA. We propose that in bacteria, DnaA oligomers of limited length and stability emerge from single highaffinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly.R egulating chromosome duplication requires precisely timed formation of nucleoprotein complexes that comprise initiator proteins bound to replication origins and that direct assembly of new replisomes (1-6). Among the best-studied examples of such nucleoprotein complexes are the origin recognition complexes (ORCs) bound to origins in budding yeast (7,8), and the complexes formed by DnaA binding to the unique origin of chromosomal replication, oriC, in Escherichia coli (6, 9). Yeast ORC subunits share structural motifs with DnaA as well as archeal Orc1 (9, 10), and all are members of the AAAϩ family of ATPases (11). This structural conservation among initiator proteins suggests the intriguing possibility that mechanisms used by all cell types to initiate DNA synthesis could be fundamentally similar (12).Examination of the binding patterns of initiator proteins to origins during the cell cycle (5,13,14) has revealed that in addition to structural similarities, there are temporal similarities in nucleoprotein complex formation at eukaryotic and prokaryotic replication origins. Yeast ORCs bind to replication origins throughout the cell cycle and recruit additional initiator proteins needed to form the prereplicative complexes (pre-RCs) that load helicase and unwind origin DNA before entry into S phase (7,8,14,15). In E. coli, a temporally similar nucleoprotein complex is formed by DnaA binding to three high-affinity (K d Ͻ 200 nM), 9-bp recognition sites (R1, R2, and R4) within oriC (Fig. 1); like yeast ORC, this binding persists throughout the majority of the cell cycle (13,16,17), except at the time of initiation, when additional initiator DnaA binds to lower-affinity (K d Ͼ 200 nM) sites in oriC (13, 18). The additional DnaA causes localized strand separation within an AT-rich, 13-mer repeat region that is adjacent to the left side of t...

BackgroundSigma factors and the alarmone ppGpp control the allocation of RNA polymerase to promoters under stressful conditions. Both ppGpp and the sigma factor σS (RpoS) are potentially subject to variability across the species Escherichia coli. To find out the extent of strain variation we measured the level of RpoS and ppGpp using 31 E. coli strains from the ECOR collection and one reference K-12 strain.ResultsNine ECORs had highly deleterious mutations in rpoS, 12 had RpoS protein up to 7-fold above that of the reference strain MG1655 and the remainder had comparable or lower levels. Strain variation was also evident in ppGpp accumulation under carbon starvation and spoT mutations were present in several low-ppGpp strains. Three relationships between RpoS and ppGpp levels were found: isolates with zero RpoS but various ppGpp levels, strains where RpoS levels were proportional to ppGpp and a third unexpected class in which RpoS was present but not proportional to ppGpp concentration. High-RpoS and high-ppGpp strains accumulated rpoS mutations under nutrient limitation, providing a source of polymorphisms.ConclusionsThe ppGpp and σS variance means that the expression of genes involved in translation, stress and other traits affected by ppGpp and/or RpoS are likely to be strain-specific and suggest that influential components of regulatory networks are frequently reset by microevolution. Different strains of E. coli have different relationships between ppGpp and RpoS levels and only some exhibit a proportionality between increasing ppGpp and RpoS levels as demonstrated for E. coli K-12.

Transfer RNAs (tRNAs) are substrates for complex enzymes, such as aminoacyl-tRNA synthetases and ribosomes, and play an essential role in translation of genetic information into protein sequences. Here we describe a general method for labeling tRNAs with fluorescent dyes, so that the activities and dynamics of the labeled tRNAs can be directly monitored by fluorescence during the ribosomal decoding process. This method makes use of the previously reported fluorescent labeling of natural tRNAs at dihydrouridine (D) positions, but extends the previous method to synthetic tRNAs by preparing tRNA transcripts and introducing D residues into transcripts with the yeast enzyme Dus1p dihydrouridine synthase. Using the unmodified transcript of Escherichia coli tRNA Pro as an example, which has U17 and U17a in the D loop, we show that Dus1p catalyzes conversion of one of these Us (mostly U17a) to D, and that the modified tRNA can be labeled with the fluorophores proflavin and rhodamine 110, with overall labeling yields comparable to those obtained with the native yeast tRNA Phe . Further, the transcript of yeast tRNA Phe , modified by Dus1p and labeled with proflavin, translocates on the ribosome at a rate similar to that of the proflavinlabeled native yeast tRNAPhe . These results demonstrate that synthetic tRNA transcripts, which may be designed to contain mutations not found in nature, can be labeled and studied. Such labeled tRNAs should have broad utility in research that involves studies of tRNA maturation, aminoacylation, and tRNA-ribosome interactions.

Most surveys for class 1 integrons are at least partly predicated on PCR screening that targets integron conserved regions. However, class 1 integrons are structurally diverse, so dependence on conserved regions may lead to missing clinically relevant examples of class 1 integrons. Here, we surveyed a commensal population of bacteria from patients in an intensive care unit to identify class 1 integrons irrespective of their structure or genetic context. We identified several examples of class 1 integrons linked to complete Tn402-like or Tn402 hybrid transposition modules and diverse insertion points with respect to the inverted repeat IRi boundary. The diversity and abundance of class 1 integrons identified are such that many novel elements seen here would not have been identified by commonly used methods, and they revealed an additional level of complexity.